Abstract

The serotonin (5-HT)2A/2C receptor antagonist ritanserin has been reported to potentiate the dopamine (DA) D2/3receptor antagonist raclopride-induced DA release in medial prefrontal cortex (mPFC) and nucleus accumbens (NAC) but not striatum (STR). Because of reciprocal interactions between 5-HT2A and 5-HT1A receptors, we tested the hypothesis that 5-HT1A receptor agonism also potentiates D2/3 receptor antagonist-induced DA release using a combination of the 5-HT1A receptor agonistR(+)-8-hydroxy-2-(di-n-propylamino)-tetralin [R(+)-8-OH-DPAT] and the D2/3 receptor antagonist S(−)-sulpiride (SUL).R(+)-8-OH-DPAT (0.05 mg/kg s.c.) potentiated low but not high dose SUL (1, 3 but not 10 or 25 mg/kg s.c.)-induced DA release in NAC, but had no effect in STR at all doses tested (1, 3, 10, and 25 mg/kg s.c.). However, R(+)-8-OH-DPAT (0.05 mg/kg s.c.) alone had no effect on basal, potentiated SUL (10 and 25 mg/kg s.c.)-induced DA release in mPFC; the effect of low dose SUL (1 and 3 mg/kg s.c.) was not tested because it alone had no effect on DA release. This potentiation was abolished by pretreatment with the 5-HT1A receptor antagonist WAY100635 (0.05 mg/kg s.c.), which alone had no effect on DA release. These results suggest that 5-HT1A receptor agonism facilitates DA release in mPFC and NAC but not STR in combination with D2 receptor antagonism.

The importance of 5-hydroxytryptamine (5-HT)1Areceptor agonist activity of antipsychotic drugs to decrease extrapyramidal symptoms (EPS) and to achieve an antipsychotic action has been proposed (Ahlenius, 1989; Millan et al., 1992). In support of this hypothesis, the combination of the dopamine (DA) D2/3 receptor antagonist raclopride with the selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) was reported to have a synergistic effect on the suppression of the conditioned avoidance response, a model for antipsychotic action (Wadenberg and Ahlenius, 1991). 5-HT1A receptor agonists also reduce haloperidol-induced catalepsy in rodents (Wadenberg, 1996), an animal model of EPS (Hoffman and Donovan, 1995). Buspirone, a 5-HT1A receptor, partial agonist, anxiolytic drug, inhibited amphetamine-induced stereotypies in rats and hyperlocomotion in mice (Gustafsson and Christensson, 1990). These behavioral effects of 5-HT1A receptor agonists may be mediated, in part, by effects on DA release in various brain regions. R(+)-8-OH-DPAT inhibited amphetamine-induced DA release in the nucleus accumbens (NAC), striatum (STR) (Ichikawa et al., 1995), and medial prefrontal cortex (mPFC) (Kuroki et al., 1996). The ability of R(+)-8-OH-DPAT to decrease stimulated DA release in the mPFC and NAC may contribute to the antipsychotic efficacy of D2 receptor antagonism. Thus, stimulation of 5-HT1A receptors attenuates stimulated DA release and consequent behaviors.

5-HT1A receptor agonists have complex effects, in a region-specific manner, on DA neurotransmission, that are not necessarily related to concomitant D2 receptor blockade. We have recently reported (J.I. and H.Y.M., in press) that R(+)-8-OH-DPAT (0.05 mg/kg s.c.) inhibited DA release in both NAC and STR produced by the following antipsychotic drugs: the 5-HT2A/D2 receptor antagonists amperozide (10 mg/kg), clozapine (20 mg/kg), and low dose risperidone (0.01 and 0.03 mg/kg). This inhibition was antagonized by the selective 5-HT1A receptor antagonist WAY100635 (n-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-n-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride) (0.05 mg/kg) (Forster et al., 1995). R(+)-8-OH-DPAT (0.05 mg/kg s.c.) pretreatment did not affect DA release in the NAC and STR produced by haloperidol (0.01, 0.03, 0.1, and 1.0 mg/kg) or high dose risperidone (0.1 and 1.0 mg/kg), respectively. These results indicate that 5-HT1Areceptor stimulation may inhibit the ability of amperozide, clozapine, and low dose risperidone, but not haloperidol and high dose risperidone, to increase DA release in the STR and NAC.

It remains to be determined whether 5-HT1Areceptor agonists affect the ability of antipsychotic drugs, including potent D2/3 receptor antagonists haloperidol and high dose risperidone, to increase DA release in the mPFC (Kuroki et al., 1999). Ritanserin, a mixed 5-HT2A/2Creceptor antagonist, has been reported to potentiate the ability of the selective D2/3 receptor antagonist raclopride to increase DA release in the mPFC and NAC but not the STR (Andersson et al., 1995). Raclopride-induced suppression of conditioned avoidance response, a model for antipsychotic activity, was potentiated by ritanserin and M100907, a selective 5-HT2Areceptor antagonist (Wadenberg et al., 1996, 1998). Because of the reciprocal interaction between 5-HT1A receptor agonists and 5-HT2A receptor antagonists (Zifa and Fillion, 1992), it is possible that 5-HT1Areceptor agonists can potentiate the ability of D2/3 receptor antagonists to increase DA release in the mPFC and perhaps the NAC. If that is the case, the concomitant use of 5-HT1A receptor agonists with D2/3 or 5-HT2A/D2 receptor antagonists may be of adjunctive value in the treatment of schizophrenia, because the ability of clozapine to increase DA release in the mPFC more than in the NAC (Kuroki et al., 1999) may contribute to its ability to decrease negative symptoms and to improve cognitive deficits by restoring cortical dopaminergic activity.

The present study was designed to test the hypothesis that a combination of the 5-HT1A receptor agonistR(+)-8-OH-DPAT and the D2/3 receptor antagonist S(−)-sulpiride (SUL), an antipsychotic drug, has a synergistic effect on DA release in the mPFC and leaves DA release unaffected in the NAC or STR. Preliminary data from this study have appeared in abstract form (Meltzer and Ichikawa, 1997).

Materials and Methods

Animals.

Male Sprague-Dawley albino rats (Zivic-Miller Laboratories, Porterville, PA) weighing 250 to 350 g were housed two per cage and maintained in a controlled 12:12-h light/dark cycle and under constant temperature at 22°C, with free access to food and water.

Surgery and Microdialysis.

Two different experiments were performed for dual probe implantation into the NAC and STR (experiment 1) or for single implantation into the mPFC (experiment 2). Rats were anesthetized with intraperitoneal injection of a combination of xylazine (6 mg/kg; Rompun, Shawnee Mission, KS) and ketamine hydrochloride (70 mg/kg, Ketaset, Fort Dodge Laboratories, Fort Dodge, IA) and mounted in a stereotaxic frame (Stoetling, Wood Dale, IL). Stainless guide cannulas (21-gauge) with a dummy probe were placed and fixed by cranioplastic cement (Plastic One, Roanoke, VA) onto the cortex dorsal both to the STR and NAC (experiment 1) or to the mPFC (experiment 2). Stereotaxic coordinates of each probe when implanted was A + 0.5, L − 4.0, V − 5.5 mm for the STR; A + 2.0, L + 1.5 to +1.7, V − 7.5 mm for the NAC; and A + 3.2, L + 1.8 at an angle of 80°, V − 5.5 mm for the mPFC, respectively, relative to bregma. The incision bar level was −3.0 mm, according to the atlas of Paxinos and Watson (1986). The length of exposed dialyzing membrane was 2 mm.

Three to 5 days after cannulation, a dialysis probe was implanted into the STR, NAC, or mPFC under slight anesthesia with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL). Rats were then housed individually overnight in a dialysis cage. After the overnight perfusion (0.2 μl/min) of the probe, the flow rate was increased to 0.8 μl/min (experiment 1) or to 0.4 μl/min (experiment 2), respectively, and then dialysate samples (24 or 12 μl, respectively) were collected every 30 min. The perfusion medium was Dulbecco's phosphate-buffered saline solution (Sigma, St. Louis, MO) including Ca2+ (138 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 0.5 mM MgCl, 1.2 mM CaCl2, pH 7.4). After stable baseline values in the dialysates were obtained, each drug or vehicle was administered to the rats. The locations of the dialysis probes were verified at the end of each experiment by brain dissection. The procedures applied in these experiments were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University in Cleveland, Ohio, where we completed the present studies.

Data Analysis.

Only results derived from healthy rats with correctly positioned dialysis probes were included in the data analysis. Mean predrug basal levels (time −60, time −30, and time 0) were designated as 100%. Repeated measure ANOVA followed by Fisher's protected least significant difference post hoc pairwise comparison procedure and one-way ANOVA were used to determine group differences (StatView 4.02 for the Macintosh). A p value <0.05 was considered significant in this study. All results are given as means ± S.E.

Results

Basal Extracellular DA Levels.

Basal extracellular DA levels (mean ± S.E. fmol/30 min, not corrected by percentage of recovery of a dialysis probe) in the dialysates were 22.8 ± 1.1 (n = 80) for the NAC, 23.1 ± 1.0 (n = 82) for the STR (experiment 1), and 1.78 ± 0.14 (n = 48) for the mPFC (experiment 2), respectively. There were no significant differences in basal extracellular DA levels in each region between treatment groups when compared. The injection of either 0.1 M tartaric acid or deionized water had no significant effect on extracellular DA levels in either region. Therefore, the combined data of the effects of both 0.1 M tartaric acid and deionized water on extracellular DA levels were used as the vehicle control for statistical analysis and graphic presentation.

In the mPFC, SUL at 10 mg/kg (F1,10 = 14.2,p < .01) and 25 mg/kg (F1,11 = 38.2, p < .01) but not 1 or 3 mg/kg (data not shown), increased extracellular DA levels, compared with vehicle controls (Fig.2). The effect of SUL was potentiated by R(+)-8-OH-DPAT (0.05 mg/kg), administered 30 min before SUL at 10 mg/kg (F1,10 = 24.1, p < .01) and 25 mg/kg (F1,10 = 39.5, p < .01), respectively, compared with the effect of each dose alone (Fig. 2). TheR(+)-8-OH-DPAT (0.05 mg/kg) potentiation of the effect of SUL at 10 mg/kg was antagonized by pretreatment with WAY100635 (0.05 mg/kg), 5 min before R(+)-8-OH-DPAT (F1,8 = 23.1, p < 0.01) (Fig.2). WAY100635 (0.05 mg/kg) had no significant effect on extracellular DA levels in the mPFC (data not shown). The effect of a combination ofR(+)-8-OH-DPAT with low dose SUL (1 and 3 mg/kg) on extracellular DA levels in the mPFC was not tested in this study because SUL alone at these doses did not significantly increase extracellular DA levels in the mPFC (data not shown).

Discussion

The major finding of the present study is that the selective 5-HT1A receptor agonist R(+)-8-OH-DPAT (0.05 mg/kg s.c.), given 30 min before the selective D2/3 receptor antagonist SUL, potentiated SUL (10 and 25 mg/kg)-induced DA release in the mPFC. These effects were completely antagonized by the selective 5-HT1Areceptor antagonist WAY100635 (0.05 mg/kg). R(+)-8-OH-DPAT (0.05 mg/kg s.c.) also potentiated the low dose SUL (1 and 3, but not 10 or 25 mg/kg)-induced DA release in the NAC but had no effect on SUL-induced DA release in the STR at all doses tested.

The reduction of serotonergic input from the dorsal raphe nucleus (DRN) may disinhibit DA activity in the ventral tegmental area (VTA). This may lead to an increase in DA release in the NAC and mPFC, to which the VTA projects DA neurons. In fact, administration of sulpiride or quinpirole, a D2/3 receptor agonist, by reverse microdialysis in the VTA has been reported to increase or decrease DA release in both the VTA and NAC, respectively (Kohl et al., 1998). Intravenous administration of low dose R(+)-8-OH-DPAT (0.002–0.032 mg/kg) increased the firing rate of DA neurons in the VTA, whereas R(+)-8-OH-DPAT (0.002–0.256 mg/kg i.v.) had no such effect in the substantia nigra zona compacta (SNC), which projects DA neurons to the STR (Arborelius et al., 1993). On the other hand, high dose R(+)-8-OH-DPAT (0.512–1.024 mg/kg i.v.) decreased the firing rate of DA neurons in both VTA and SNC, an effect antagonized by the D2/3 receptor antagonist raclopride (Arborelius et al., 1993). Thus, low and high doseR(+)-8-OH-DPAT may activate or suppress DA activity in the VTA, respectively. In accord with these electrophysiological studies, low dose (±)-8-OH-DPAT (0.05 mg/kg s.c.) but not high dose (0.5 mg/kg s.c.), increased DA release in the VTA (Chen and Reith, 1995).R(+)-8-OH-DPAT (0.025 mg/kg s.c.; Arborelius et al., 1993) and (±)-8-OH-DPAT (0.03 mg/kg s.c.; Tanda et al., 1994), in low doses, increased DA release in the mPFC, whereas (±)-8-OH-DPAT (0.225 mg/kg) and R(+)-8-OH-DPAT (0.2 mg/kg s.c.), in high doses, decreased DA release in the STR (Rasmusson et al., 1994) and both the STR and NAC (J.I. and H.Y.M., in press), respectively. However,R(+)-8-OH-DPAT (0.05 mg/kg s.c.) had no effect on basal DA release in the STR, NAC, or mPFC in the present study. Because of the ability of (±)-8-OH-DPAT (0.225 mg/kg; Rasmusson et al., 1994) andR(+)-8-OH-DPAT (0.1 and 0.2 mg/kg s.c.; Ishii et al., 1999) in high doses to increase DA release in the mPFC, a dose of 0.05 mg/kg may be subthreshold under the conditions of the present study.

Direct application of 8-OH-DPAT into the DRN has been reported to decrease DA release in the NAC (Yoshimoto and McBride, 1992), whereas electrical stimulation of the DRN has been reported to increase and decrease DA release in the NAC and STR, respectively (De Deurwaerdère et al., 1998). Reduction or activation of serotonergic activity in the DRN leads to an increase or decrease, respectively, in DA release in the terminal regions of the VTA, as discussed above. If this conclusion is correct, direct application of 8-OH-DPAT into the DRN should increase DA release in the NAC, whereas electrical stimulation of the DRN should decrease DA release in the NAC. However, the results reported by Yoshimoto and McBride (1992) andDe Deurwaerdère et al. (1998) are entirely the opposite of these predictions. Furthermore, the mixed 5-HT2A/2Creceptor antagonist ritanserin, which acutely increases the firing rate of DA neurons in the VTA and SNC (Ugedo et al., 1989), has been reported not to increase DA release in the STR, NAC, or mPFC (Andersson et al., 1995). 5-HT1A receptors may be functional in the STR and perhaps the NAC, despite their sparse distribution (Pompeiano et al., 1992; Khawaja et al., 1995) because inactivation of 5-HT1A autoreceptors by pertussis toxin in the DRN did not affect the ability of 8-OH-DPAT to decrease striatal 5-HT release (Romero et al., 1994), and administration ofR(+)-8-OH-DPAT by reverse microdialysis decreased DA release in the STR and NAC (Nomikos et al., 1996). Taken together, the effects of R(+)-8-OH-DPAT on basal and SUL-induced DA release in the STR, NAC, and mPFC are unlikely to be mediated solely by disinhibition of dopaminergic activity in the VAT due to reduction of serotonergic input from the DRN.

(±)-8-OH-DPAT (0.05 and 0.5 mg/kg s.c.) has been reported to potentiate the local perfusion of sulpiride (10 μM)-induced DA release in the VTA (Chen and Reith, 1995). This is consistent with the present results. SUL (10 and 25 mg/kg) produced comparable increases in DA release in the NAC. These doses may produce maximum DA release in the NAC, presumably due to saturation of the effect of D2 autoreceptor blockade on DA release. In fact,R(+)-8-OH-DPAT (0.05 mg/kg s.c.) potentiated only the lower dose SUL (1 and 3 mg/kg)-induced DA release in the NAC. On the other hand, the ability of SUL to increase striatal DA release at all doses (1, 3, 10, and 25 mg/kg) was not affected by R(+)-8-OH-DPAT (0.05 mg/kg s.c.). Differences in the effects ofR(+)-8-OH-DPAT on the ability of SUL to increase DA release in the STR, NAC, and mPFC may be due, at least in part, to the comparable affinity of SUL for D2 and D3 receptors (Seeman and Van Tol, 1994). D3 receptor binding sites are distributed with highest densities in the islands of Calleja, olfactory tubercles, and NAC, and are distributed with low density in the STR and mPFC of rodent brains (Bancroft et al., 1998). An inhibitory effect of D3 receptors on DA release, independent of D2 receptors, has been suggested (Rivet et al., 1994; Gobert et al., 1996; Fink-Jensen et al., 1998). Therefore, greater blockade of D3 receptors in the NAC compared with the STR may contribute to the ability of SUL to increase DA release, although further studies are needed to test this hypothesis.

Sulpiride is an antipsychotic drug with low liability of producing EPS and has antidepressant effects (Willner, 1997). The results reported here suggest that 5-HT1A receptor agonists combined with sulpiride or other D2/3 receptor antagonists may be useful to reduce negative symptoms by facilitating prefrontal cortical DA release, if this facilitation leads to a decrease in negative symptoms and improves cognitive deficits in schizophrenia (Davis et al., 1991). Interestingly, low- but not high-dose SUL-induced DA release in the NAC was potentiated byR(+)-8-OH-DPAT. These results suggest that high dose SUL, which produces full blockade of the NAC D2receptors, together with a 5-HT1A receptor agonist may produce greater facilitation of dopaminergic transmission in the mPFC compared with the NAC, whereas the combination of low dose SUL and 5-HT1A receptor agonism may facilitate dopaminergic transmission in both regions because of weak D2 receptor blockade in the NAC. The absence of synergistic effects of SUL and R(+)-8-OH-DPAT on striatal DA release indicate that 5-HT1A receptor agonists are unlikely to affect D2 receptor antagonist-induced DA release in the STR. This raises the possibility that DA release in the NAC produced by low dose SUL is mediated by blockade of D3 receptors. This suggestion supports previous evidence for the importance of D3 receptors in the treatment of schizophrenia (Sokoloff et al., 1990; Schwartz et al., 1998) and depression (Willner, 1997). Facilitation of DA neurotransmission in the NAC by D3 receptor antagonism may be clinically relevant in mood and addictive disorders.

In summary, the present in vivo microdialysis studies support the hypothesis that, similar to the effect of 5-HT2Areceptor antagonism, 5-HT1A receptor agonism and D2/3 receptor antagonism have a synergistic effect on DA release in the mPFC and NAC but not the STR. These results suggest that 5-HT1A receptor agonist properties of antipsychotic drugs, regardless of whether they are added or intrinsic, e.g., ziprasidone (Seeger et al., 1995) and S16924 (Millan et al., 1998), facilitate therapeutic efficacy by increasing prefrontal cortical DA release. The present studies also support the important role of D3 receptors in the regulation of DA release in the NAC, which is relevant to antipsychotic action and possibly antidepressant and antiaddictive actions.

Acknowledgments

We are grateful to Michael T. Kitchen for excellent laboratory assistance.

Footnotes

↵1 This work was supported in part by U.S. Public Health Services Grant MH41684. Preliminary data from this study have been reported in abstract form at the annual meeting of the Society for Neuroscience, 1997.

(1998) Opposite change of in vivo dopamine release in the rat nucleus accumbens and striatum that follows electrical stimulation of dorsal raphe nucleus: Role of 5-HT3 receptors.J Neurosci18:6528–6538.

(1996) Enhancement of antipsychotic-like properties of the dopamine D2 receptor antagonist, raclopride, by the additional treatment with the 5-HT2 receptor blocking agent, ritanserin, in the rat.Eur J Neuropsychopharmacol6:305–310.